Summary Interleukin-4 is a prototypic immunoregulatory cytokine. It is the central regulator of allergic inflammatory responses, controlling the polarization of naive CD4 T cells to the Th2 phenotype and Ig class switching to IgE. The Cytokine Biology Unit has characterized the signaling mechanisms utilized by the IL-4 receptor. It has shown that activation of the latent transcription factor, Stat 6, controls both TH2 polarization and IgE class switching and that GATA3 and Stat5 play essential roles in the acquisition of IL-4 producing capacity. Unit scientists have carried out genome-wide analyses of histone modification and transcription factor binding in each of the various Th lineages to gain insight into the processes underlying Th differentiation. They have identified a large number of sites to which GATA3 binds and sites for other transcription factors that bind in close proximity suggesting that these transcription factors act together with GATA3 in mediating transcriptional regulation. This information has provided important insights into the genetic regulation of Th2 differentiation. Our analysis has shown that GATA3 mediates specific repressive functions in lineages other than Th2 cells and thus contribute to shaping the pattern of gene expression in various cell populations. Proteomic studies using mass spectrometry has identified several GATA3 partners of which three appear particularly interesting. They are BCL11b, Ikaros, and E4BP4 (NF-IL-3). Studies are in progress to identify those sites to which both GATA3 and each bind and to determine the consequences of knocking down or knocking out these transcription factors on GATA3 functions. Unit scientists have now been shown that T cell receptor-mediated activation of the ERK signaling pathway blocks Th2 differentiation by preventing transcription of GATA3 and desensitizing the IL-2 receptor. Strong TCR-mediated signals stimulate ERK phosphorylation, thereby preventing TH2 differentiation and accounting for poor Th2 differentiation at high antigen concentration. Low concentrations of antigen, which activate ERK only weakly, are permissive for early IL-4 production and TH2 differentiation. The targets of ERK action that mediate suppression of GATA3 transcription are under active study as are the transcription factors that mediate TCR induced GATA3 transcription. We have confirmed that Notch signaling participates in Th2 priming mainly at low TCR signal strength and have shown that it does so by regulating both early IL-2 production and the sensitivity of the IL-4 receptor to IL-4 by increasing the level Stat-6 phosphorylation to a standard challenge of IL-4. The latter is due to repression of PTP1B expression by Notch signalling. Knocking down PTP1b expression in CD4 T cells enhances their Th2 differentiation in the absence of Notch. In addition, the defect in Th2 priming in mice with defects in the Notch pathway can be rescued by the addition of IL-2. Thus repressing PTP1b and enhancing IL-2 largely rescue the Notch defect in Th2 priming. In the course of studying control of The differentiation by Notch, the discovery was made that deletion of presinilin (PS), a key component of gamma-secretase, which is essential for Notch function, leads to an unexpected result. The PS deficeint cells can efficeintlly differentiate into IL-4-producers when stimulated strongly through the TCR. They are able to continue to signal through the IL-2 receptor/ Stat5 pathway, implyng that PS eiither is downstream of ERK in desnesitizing the IL-2 recpetor or works in parallel with ERK. Recent studies have revealed that one can manipulate the pattern of IL-4 responsivenss in various target cells through mutations in key regions of IL-4 that increase its affinity for either the gamma common chian or for IL-13R alpha, thus making IL-4 a largely regulatory of a largely effector cytokines and making more tractable for in vivo use. Such mutants were prepared by our collaborator, KC Garcia, and were shown by us to have distinctive functions on various target cells, particularly measuring differential STAT6 phopshorylation. We also modeled these effects using a Matlab script that provided us with important predictive informattion on the the relationship between level of receptor expression, affinity of receptor and the number of assembled complexes. LI scientists have also developed new insights into the cytokine-stimulated production of IL-13 by basophils and mast cells. IL-3 activating STAT5 collaborates with IL-33 to activate mast cells IL-13 production; by contrast basophil IL-13 production can be stimulated most efficeintly by IL-3 plus IL-18. Laboratory scientists have developed a series of indicator mice. Particularly valuable are those that reflect the expression of IL-4, IL-13 and TSLP. These indicator mice were made through the introduction of a bacterial artificial chromosomes in which the AM-Cyan gene reported IL-4, destabilized DS-Red has reported IL-13 and ZS-green reported TSLP expression. These mice have proved to be excellent reporters of cytokine production in Schistosomiasis infection and their value in other infections is being studied.